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J. Biol. Chem., Vol. 276, Issue 41, 37779-37786, October 12, 2001

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Constitutively Active µ-Opioid Receptors Inhibit Adenylyl Cyclase Activity in Intact Cells and Activate G-proteins Differently than the Agonist [D-Ala²,N-MePhe⁴,Gly-ol⁵]Enkephalin^*

Jing-Gen Liu, Michael B. Ruckle, and Paul L. Prather

From the Department of Pharmacology and Toxicology, College of Medicine, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205

The most convincing evidence demonstrating constitutive activation of µ-opioid receptors is the observation that putative inverse agonists decrease basal G-protein activity in membrane preparations. However, it is not clear whether constitutively active receptors in isolated membranes have any physiological relevance in intact cells. GH₃ cells expressing µ-opioid receptors (GH₃MOR) exhibit higher basal G-protein activity and lower basal cAMP levels than wild-type GH₃ cells, indicative of constitutively active receptors. This study determined whether alkylation of µ-opioid receptors by the irreversible antagonist -funaltrexamine would decrease spontaneous receptor activity in intact cells, revealing constitutive activity. GH₃MOR cells were pretreated with increasing concentrations of -funaltrexamine followed by functional testing after removal of unbound drug. -Funaltrexamine pretreatment produced a concentration-dependent decrease in µ-opioid receptor binding with an IC₅₀ of 0.98 nM and an E_max of 77%. Similar concentrations of -funaltrexamine pretreatment produced a half-maximal reduction in basal [³⁵S]GTPS binding, a decrease in basal photolabeling of G-proteins with azidoanilido-[-³²P]GTP, and an increase in basal adenylyl cyclase activity in intact cells. Therefore, µ-opioid receptors are constitutively active in intact cells, producing stimulation of G-proteins and inhibition of adenylyl cyclase. Importantly, photolabeling of G-subunits with azidoanilido-[-³²P]GTP demonstrated that constitutively active µ-opioid receptors activate individual G-proteins differently than the agonist [D-Ala²,N-MePhe⁴,Gly-ol⁵]enkephalin.

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